Difference between revisions of "Part:BBa R0082:Experience"

<I>Edinburgh iGEM 2009</I>

<I>Edinburgh iGEM 2009</I>

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We tested this promoter for use in our landmine-detection system. Our plan was to use this promoter together with a computationally designed TNT receptor and the hybrid Trz signal transduction protein, which has the EnvZ kinase domain, hence phosphorylates OmpR, activating this promoter, in the presence of TNT. To check that this promoter was working correctly, we attached it to GFP and assayed its activity in ''E. coli'' TOP10 cells as described on the [https://parts.igem.org/Measurement Registry Measurement Page]. (We also made a ''lacZ'' reporter version, BBa_K216010, so that we could do Miller assays as a backup, but at the time of writing we have not obtained good quantitative results from this). To induce the promoter, we used procaine, which activates the EnvZ kinase. Our results indicated that the promoter is activated by increasing levels of procaine from 0 to 15 mM, but there is very high basal activity under the conditions used, even in the absence of procaine. We also made an ''envZ'' mutant host strain using the KEIO collection of ''E. coli'' mutants, but at the time of writing, we have not yet repeated the tests using this host.

We tested this promoter for use in our landmine-detection system. Our plan was to use this promoter together with a computationally designed TNT receptor and the hybrid Trz signal transduction protein, which has the EnvZ kinase domain, hence phosphorylates OmpR, activating this promoter, in the presence of TNT. To check that this promoter was working correctly, we attached it to GFP and assayed its activity in ''E. coli'' TOP10 cells as described on the [https://parts.igem.org/Measurement Registry Measurement Page]. (We also made a ''lacZ'' reporter version, BBa_K216010, so that we could do Miller assays as a backup, but at the time of writing we have not obtained good quantitative results from this). To induce the promoter, we used procaine, which activates the EnvZ kinase. Our results indicated that the promoter is activated by increasing levels of procaine from 0 to 15 mM, but there is very high basal activity under the conditions used, even in the absence of procaine. We also made an ''envZ'' mutant host strain using the KEIO collection of ''E. coli'' mutants, but at the time of writing, we have not yet repeated the tests using this host.

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